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Series GSE138169 Query DataSets for GSE138169
Status Public on Jun 01, 2020
Title Engineered receptors for human cytomegalovirus that are orthogonal to normal human biology
Organism Homo sapiens
Experiment type Other
Summary A trimeric glycoprotein complex on the surface of human cytomegalovirus (CMV) binds to platelet-derived growth factor (PDGF) receptor α (PDGFRα) to mediate host cell recognition and fusion of the viral and cellular membranes. Soluble PDGFRα potently neutralizes CMV in tissue culture, and its potential use as an antiviral therapeutic has the benefit that any escape mutants will likely be attenuated. However, PDGFRα binds multiple PDGF ligands in the human body as part of developmental programs in embryogenesis and continuing through adulthood. Any therapies with soluble receptor therefore come with serious efficacy and safety concerns, especially for the treatment of congenital CMV. Soluble virus receptors that are orthogonal to human biology would reduce safety and efficacy concerns. This engineering problem is solved by deep mutational scanning on the D2-D3 domains of PDGFRα to identify variants that maintain interactions with the CMV glycoprotein trimer in the presence of competing PDGF ligands.
Overall design Site-saturation mutagenesis was focused on the D2-D3 domains (a.a. D123-E311) of human PDGFRα, thereby creating a library of variants encompassing all possible single amino acid substitutions within the structural elements that form the PDGF binding site. The PDGFRα plasmid library, bearing an extracellular c-myc epitope tag for detection of surface expression, was transfected in to Expi293F cells; this line is a suspension culture derivative of HEK 293. The transfected culture was incubated with medium from cells expressing soluble CMV trimer (glycoproteins gH-gL-gO, where gO was genetically fused at its C-terminus to superfolder GFP), and co-stained with a fluorescent anti-myc antibody. Trimer binding was readily detected based on GFP fluorescence, and was proportional to PDGFRα expression levels. In the presence of competing PDGF ligands (25 nM each of PDGF-AA, -BB, -AB, and -CC), a subset of variants in the library maintained high CMV trimer binding. These variants were collected by fluorescence-activated cell sorting (FACS). In addition, cells expressing PDGFRα but with low CMV trimer binding were also collected, so that the effects of mutations on receptor expression versus trimer binding could be teased apart. RNA was extracted from the collected cells, reverse transcribed with a high fidelity polymerase, and relevant coding regions were PCR amplified for sequencing. Following Illumina sequencing, the frequencies of PDGFRα variants in the transcripts were compared to their frequencies in the naive plasmid library, such that the enrichment or depletion of all sequence variants was determined.
Contributor(s) Procko E
Citation(s) 32559251
Submission date Sep 30, 2019
Last update date Jul 20, 2020
Contact name Erik Procko
Phone 217-300-1454
Organization name University of Illinois
Department Biochemistry
Lab RAL 318G
Street address 601 S Goodwin Ave
City Urbana
State/province IL
ZIP/Postal code 61801
Country USA
Platforms (1)
GPL24676 Illumina NovaSeq 6000 (Homo sapiens)
Samples (5)
GSM4101198 Naive PDGFRA-D2D3 library
GSM4101199 UL74-High Replicate1
GSM4101200 UL74-High Replicate2
BioProject PRJNA574965
SRA SRP223712

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Supplementary file Size Download File type/resource
GSE138169_enrichment_ratios_PDGFRA_gHgLgO.xlsx 261.4 Kb (ftp)(http) XLSX
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Raw data are available in SRA
Processed data are available on Series record

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